Biomedical subjects
Publications and source records attributed to Ruth N Collins.
Understanding the molecular mechanisms that control the architecture of organelles is an area of intense study. In this issue of Cell, Voeltz et al. (2006) report that two membrane proteins, Rtn4a/NogoA and DP1/Yop1p, are responsible for the generation of tubular morphology in the endoplasmic reticulum (ER). The unusual membrane topology of these proteins may directly contribute to ER curvature.
A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising multiple Rab orthologs from diverse species. We compared the locations of individual yeast Rab proteins with their localizations following ectopic expression in mammalian cells. Our results suggest that green fluorescent protein-tagged Rab proteins maintain localizations across large evolutionary distances and that the major known player in the Rab localization pathway, mammalian Rab-GDI, is able to function in yeast. These findings enable us to provide insight into novel gene functions and classify the uncharacterized Rab proteins Ypt10p (YBR264C) as being involved in endocytic function and Ypt11p (YNL304W) as being localized to the endoplasmic reticulum, where we demonstrate it is required for organelle inheritance.
The activation of Rab GTPases is a critical focal point of membrane trafficking events in eukaryotic cells; however, the cellular mechanisms that spatially and temporally regulate this process are poorly understood. Here, we identify a null allele of ELP1 as a suppressor of a mutant in a Rab guanine nucleotide exchange factor Sec2p. Elp1p was previously thought to be involved in transcription elongation as part of the Elongator complex. We show that elp1Delta suppression of sec2(ts) is not a result of reduced transcriptional elongation and that Elp1p physically associates with Sec2p. The Sec2p interaction domain of Elp1p is necessary for both Elp1p function and for the polarized localization of Sec2p. Mutations in human Elp1p (IKAP) are a known cause of familial dysautonomia (FD). Our results raise the possibility that regulation of polarized exocytosis is an evolutionarily conserved function of the entire Elongator complex and that FD results from a dysregulation of neuronal exocytosis.
Yeast Yip1p is a member of a conserved family of transmembrane proteins that interact with Rab GTPases. Previous studies also have indicated a role for Yip1p in the biogenesis of endoplasmic reticulum (ER)-derived COPII transport vesicles. In this report, we describe the identification and characterization of the uncharacterized open reading frame YER074W-A as a novel multicopy suppressor of the thermosensitive yip1-4 strain. We have termed this gene Yip One Suppressor 1 (YOS1). Yos1p is essential for growth and for function of the secretory pathway; depletion or inactivation of Yos1p blocks transport between the ER and the Golgi complex. YOS1 encodes an integral membrane protein of 87 amino acids that is conserved in eukaryotes. Yos1p localizes to ER and Golgi membranes and is efficiently packaged into ER-derived COPII transport vesicles. Yos1p associates with Yip1p and Yif1p, indicating Yos1p is a novel subunit of the Yip1p-Yif1p complex.
The continuing explosion of sequencing data has inspired a corresponding effort in the annotation and classification of protein families. Within a particular protein family, however, individual members may have distinct functions, although they share a common fold and broadly defined physiological role. Rab GTPases are the largest subfamily of the Ras superfamily, yet from early in their discovery, it was apparent that each Rab protein has a unique subcellular localization and regulates a particular stage(s) membrane traffic. To gain insight into the contribution of individual residues to unique protein functions a general strategy is outlined. This method should allow the cell and molecular biologist with no specialist expertise to implement an algorithm that makes use of a combination of experimental and phylogenetic data. The algorithm is applicable to the analysis of any protein domain and here is illustrated with the analysis of residues contributing to the individual functions of a pair of Rab GTPases.
Researchers looking to solve biological problems have access to enormous amounts of sequence information and the desktop computational infrastructure to personally interrogate and analyze large datasets. Many powerful bioinformatics tools are available online; however, this discourages the customized analysis of data that is necessary for the experimental scientist to make maximally effective use of the information. In addition, a customized environment facilitates the critical evaluation of bioinformatic methods. This chapter presents a protocol developed to aid in classification of subfamilies and subclasses of a superfamily using the personal desktop computer. The visual representation of the qualitative and quantitative results of data analyses is also considered. The examples are focused on Rab GTPases but are more widely applicable to the classification of any given protein family.
The YIP1 family of proteins is an intriguing collection of small membrane proteins with critical roles in membrane traffic. Although their mode of action is unknown, they are receiving attention as participants in vesicle biogenesis, and as factors that may mediate the association of Rab proteins with membranes. Yeast YIP1 is an essential gene and can be fully complemented by its human counterpart-suggesting that the essential function of Yip1p is evolutionarily conserved. This chapter presents methods for the cell biological and genetic analysis of Yip1p and other YIP1 family members in the yeast Saccharomyces cerevisiae.
Rab GTPases are crucial regulators of organelle biogenesis, maintenance, and transport. Multiple Rabs are expressed in all cells, and each is localized to a distinct set of organelles, but little is known regarding the mechanisms by which Rabs are targeted to their resident organelles. Integral membrane proteins have been postulated to serve as receptors that recruit Rabs from the cytosol in a complex with the Rab chaperone, GDI, to facilitate the dissociation of Rab and GDI, hence facilitating loading of Rabs on membranes. We show here that the yeast (Saccharomyces cerevisiae) Golgi Rab GTPase Ypt1p can be copurified with the integral membrane protein Yip3p from detergent cell extracts. In addition, a member of the highly conserved reticulon protein family, Rtn1p, is also associated with Yip3p in vivo. However, Ypt1p did not copurify with Rtn1p, indicating that Yip3p is a component of at least two different protein complexes. Yip3p and Rtn1p are only partially colocalized in cells, with Yip3p localized predominantly to the Golgi and secondarily to the endoplasmic reticulum, whereas Rtn1p is localized predominantly to the endoplasmic reticulum and secondarily to the Golgi. Surprisingly, the intracellular localization of Rabs was not perturbed in yip3Delta or rtn1Delta mutants, suggesting that these proteins do not play a role in targeting Rabs to intracellular membranes. These data indicate that Yip3p may have multiple functions and that its interaction with Rabs is not critical for their recruitment to organelle membranes.
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Yip1p is the first identified Rab-interacting membrane protein and the founder member of the YIP1 family, with both orthologs and paralogs found in all eukaryotic genomes. The exact role of Yip1p is unclear; YIP1 is an essential gene and defective alleles severely disrupt membrane transport and inhibit ER vesicle budding. Yip1p has the ability to physically interact with Rab proteins and the nature of this interaction has led to suggestions that Yip1p may function in the process by which Rab proteins translocate between cytosol and membranes. In this study we have investigated the physiological requirements for Yip1p action. Yip1p function requires Rab-GDI and Rab proteins, and several mutations that abrogate Yip1p function lack Rab-interacting capability. We have previously shown that Yip1p in detergent extracts has the capability to physically interact with Rab proteins in a promiscuous manner; however, a genetic analysis that covers every yeast Rab reveals that the Rab requirement in vivo is exclusively confined to a subset of Rab proteins that are localized to the Golgi apparatus.
Yeast Ypt1p-interacting protein (Yip1p) belongs to a conserved family of transmembrane proteins that interact with Rab GTPases. We encountered Yip1p as a constituent of ER-derived transport vesicles, leading us to hypothesize a direct role for this protein in transport through the early secretory pathway. Using a cell-free assay that recapitulates protein transport from the ER to the Golgi complex, we find that affinity-purified antibodies directed against the hydrophilic amino terminus of Yip1p potently inhibit transport. Surprisingly, inhibition is specific to the COPII-dependent budding stage. In support of this in vitro observation, strains bearing the temperature-sensitive yip1-4 allele accumulate ER membranes at a nonpermissive temperature, with no apparent accumulation of vesicle intermediates. Genetic interaction analyses of the yip1-4 mutation corroborate a function in ER budding. Finally, ordering experiments show that preincubation of ER membranes with COPII proteins decreases sensitivity to anti-Yip1p antibodies, indicating an early requirement for Yip1p in vesicle formation. We propose that Yip1p has a previously unappreciated role in COPII vesicle biogenesis.
The majority of Rab proteins are posttranslationally modified with two geranylgeranyl lipid moieties that enable their stable association with membranes. In this study, we present evidence to demonstrate that there is a specific lipid requirement for Rab protein localization and function. Substitution of different prenyl anchors on Rab GTPases does not lead to correct function. In the case of YPT1 and SEC4, two essential Rab genes in Saccharomyces cerevisiae, alternative lipid tails cannot support life when present as the sole source of YPT1 and SEC4. Furthermore, our data suggest that double geranyl-geranyl groups are required for Rab proteins to correctly localize to their characteristic organelle membrane. We have identified a factor, Yip1p that specifically binds the di-geranylgeranylated Rab and does not interact with mono-prenylated Rab proteins. This is the first demonstration that the double prenylation modification of Rab proteins is an important feature in the function of this small GTPase family and adds specific prenylation to the already known determinants of Rab localization.
The formation of coated vesicles is a fundamental step in many intracellular trafficking pathways. COPI and clathrin represent two important and distinct sets of vesicle coating machinery, involved primarily in mediating intra-Golgi and endocytic transport, respectively. Here we identify an important functional region at the carboxyl terminus of the gamma subunit of the COPI complex (gammaCOP) and describe the X-ray crystal structure of this domain at 2.3 A resolution. This domain of gammaCOP exhibits unexpected structural similarity to the carboxyl-terminal appendage domains of the alpha and beta subunits of the AP2 adaptor proteins, integral components of clathrin-coated vesicles. The remarkable structural conservation exhibited by the gammaCOP appendage domain, coupled with functional data and primary sequence analysis, supports a model of COPI function with significant structural and mechanistic parallels to vesicular transport by the clathrin/AP2 system.
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The Rab GTPases are key regulators of membrane traffic. Yip1p is a membrane protein of unknown function that has been reported to interact with the Rabs Ypt1p and Ypt31p. In this study we identify Yif1p, and two unknown open reading frames, Ygl198p and Ygl161p, which we term Yip4p and Yip5p, as Yip1p-related sequences. We demonstrate that the Yip1p-related proteins possess several features: (i) they have a common overall domain topology, (ii) they are capable of biochemical interaction with a variety of Rab proteins in a manner dependent on C-terminal prenylation, and (iii) they share an ability to physically associate with other members of the YIP1 family.
The regulation of membrane traffic involves the Rab family of Ras-related GTPases, of which there are a total of 11 members in the yeast Saccharomyces cerevisiae. Previous work has identified PRA1 as a dual prenylated Rab GTPase and VAMP2 interacting protein [Martinic et al. (1999) J. Biol. Chem. 272, 26991-26998]. In this study we demonstrate that the yeast counterpart of PRA1 interacts with Rab proteins and with Yip1p, a membrane protein of unknown function that has been reported to interact specifically with the Rab proteins Ypt1p and Ypt31p. Yeast Pra1p/Yip3p is a factor capable of biochemical interaction with a panel of different Rab proteins and does not show in vitro specificity for any particular Rab. The interactions between Pra1p/Yip3p and Rab proteins are dependent on the presence of the Rab protein C-terminal cysteines and require C-terminal prenylation.
Myosins are molecular motors that exert force against actin filaments. One widely conserved myosin class, the myosin-Vs, recruits organelles to polarized sites in animal and fungal cells. However, it has been unclear whether myosin-Vs actively transport organelles, and whether the recently challenged lever arm model developed for muscle myosin applies to myosin-Vs. Here we demonstrate in living, intact yeast that secretory vesicles move rapidly toward their site of exocytosis. The maximal speed varies linearly over a wide range of lever arm lengths genetically engineered into the myosin-V heavy chain encoded by the MYO2 gene. Thus, secretory vesicle polarization is achieved through active transport by a myosin-V, and the motor mechanism is consistent with the lever arm model.
Our understanding of the mechanisms governing of Rab and Arf protein function has exploded in recent years with a convergence of information from model genetic organisms, biochemical studies, cell biological observations and protein structural information. However, the list of known Rab and Arf interacting factors still remains small relative to the number of these small GTPases that have been identified through complete genomic sequencing. It can be anticipated that the factors listed and discussed in this review probably represent a small fraction of the Rab and Arf accessory molecules that remain to be discovered. The identification of regulators and accessory molecules for the Rab and Arf families has allowed investigators to elaborate themes and develop a framework for a mechanistic understanding of these proteins. The themes are highlighted in this review, which aims to concentrate on Rab and Arf function in exocytosis.